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Biomedical subjects

K Omori

Publications and source records attributed to K Omori.

At least 19 recordsLinked to original sources
PubMed2006

Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization.

Type I secretion system (TISS) of Gram-negative bacteria permits proteins to be secreted directly from the cytoplasm to the external medium by a single, energy-coupled step. To examine whether this system can be used as an extracellular production system of recombinant proteins, Escherichia coli alkaline phosphatase (AP) was fused to a C-terminal region of Pseudomonas sp. MIS38 lipase (PML) and examined for secretion using the E.coli cells carrying the heterologous TISS. PML is one of the passenger proteins of TISS and contains 12 repetitive sequences and a secretion signal at the C-terminal region. The fusion protein was efficiently secreted to the extracellular medium, while AP was not secreted at all, indicating that the secretion of AP is promoted by a secretion signal of PML. The repetitive sequences were not so important for secretion of the fusion protein, because the secretion level of the fusion protein containing entire repeats ( approximately 10 mg/l culture) was only 2-fold higher than that of the fusion protein without repeats. The fusion protein purified from the culture supernatant existed as a homodimer, like AP, and was indistinguishable from AP in enzymatic properties and stability.

Alkaline Phosphatase↗
PubMed2006

Etiology and prognostic significance of severe uremic pruritus in chronic hemodialysis patients.

Although uremia is well known as the most common cause of pruritus, the mechanisms of pruritus in chronic hemodialysis patients remain unclear. The purpose was to characterize uremic pruritus in more detail and to investigate whether severe pruritus is a marker for poor prognosis. A total of 1773 adult hemodialysis patients were studied. A questionnaire was given to each patient to assess the intensity and frequency, as well as pruritus-related sleep disturbance. We analyzed the relationship between clinical and laboratory data and the severity of pruritus in hemodialysis patients and followed them for 24 months prospectively. In total, 453 patients had severe pruritus with a visual analogue scale (VAS) score more than or equal to 7.0. Among them, more than 70% complained of sleep disturbance, whereas the majority of patients with a VAS score of less than 7.0 had no sleep disturbance. Male gender, high levels of blood urea nitrogen, beta2-microglobulin (beta2MG), hypercalcemia, and hyperphosphatemia were identified as independent risk factors for the development of severe pruritus, whereas a low level of calcium and intact-parathyroid hormone were associated with reduced risk. During the follow-up, 171 (9.64%) patients died. The prognosis of patients with severe pruritus was significantly worse than the others. Moreover, severe pruritus was independently associated with death even after adjusting for other clinical factors including diabetes mellitus, age, beta2MG, and albumin. Severe uremic pruritus caused by multiple factors, not only affects the quality of life but may also be associated with poor outcome in chronic hemodialysis patients.

Aged↗
PubMed2005

Increased expression of cyclooxygenase-2 in human pancreatic islets treated with high glucose or ligands of the advanced glycation endproduct-specific receptor (AGER), and in islets from diabetic mice.

AIMS/HYPOTHESIS: The cyclooxygenase-2 (PTGS2, previously known as COX2) enzyme and its products, such as prostaglandin E(2) (PGE(2)), have been implicated in the pathogenesis of several inflammatory diseases including islet dysfunction under diabetic conditions. In this study we evaluated whether diabetic conditions in vitro, such as high-glucose (HG) culture or AGE, or in vivo in animal models of diabetes can induce PTGS2 expression and activity in pancreatic islets. MATERIALS AND METHODS: Isolated human pancreatic islets were treated for 24 h with HG (25 mmol/l) or with S100b (5 mg/l), a specific ligand for the AGE-specific receptor. PTGS2 and cyclooxygenase-1 (PTGS1, previously known as COX1) mRNA, protein expression and product PGE(2) were analysed by RT-PCR, Western blots and specific enzyme immunoassay respectively. Islet PTGS2 production in animal models was assessed by immunofluorescence. RESULTS: Treatment of human pancreatic islets with HG and S100b led to a three-five-fold induction of PTGS2 mRNA (p<0.001). PTGS2 protein and its product PGE(2) (351.4+/-13.05 fg/ml vs control 39.4+/-0.11 fg/ml) were also increased (p<0.001). Pretreatment with specific inhibitors demonstrated the involvement of protein kinase C and oxidant stress in S100b- and HG-induced PTGS2 expression. However, insulin secretion was not significantly altered by S100b. Double immunofluorescent staining showed increased PTGS2 production in pancreatic islets from diabetic mice relative to corresponding controls. CONCLUSION/INTERPRETATION: These results show for the first time that diabetes as well as diabetic conditions such as AGE and HG in vitro can directly upregulate the expression of the inflammatory PTGS2 gene in pancreatic islets. This might contribute to the pathogenesis of islet dysfunction in diabetes.

Animals↗
PubMed2005

Multipotent progenitor cells isolated from adult human pancreatic tissue.

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.

Adult↗
PubMed2005

Inhibition of p38 mitogen-activated protein kinase protects human islets from cryoinjury and improves the yield, viability, and quality of frozen-thawed islets.

The development of an optimal islet cryopreservation method will permit transplantation of islets from multiple donors in a single procedure and contribute to alleviation of the islet shortage. In this study, we have improved human islet cryopreservation methods under serum-free conditions using an intracellular-based islet cryopreservation solution (ICS), especially supplemented with a p38 pathway inhibitor (p38IH) to suppress p38 mitogen-activated protein kinase (MAPK) activation. Three different solutions were compared for freezing and thawing of human islets (1) conventional RPMI1640 medium, (2) ICS, and (3) ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Islet cryopreservation with ICS-p38IH significantly improved islet recovery, viability, and quality after thawing of cryopreserved islets. This improvement may allow the use of cryopreserved islets in clinical islet transplantation.

Cell Culture Techniques↗
PubMed2005

Maxacalcitol therapy decreases circulating osteoprotegerin levels in dialysis patients with secondary hyperparathyroidism.

BACKGROUND: Osteoprotegerin is a natural glycoprotein which plays a critical role in osteoclast physiology. Elevated levels of circulating osteoprotegerin may account for the development of bone and mineral metabolic abnormalities in uremia. Little is known about the effects of vitamin D therapy on the circulating osteoprotegerin levels in dialysis patients. PATIENTS AND METHODS: Fifty chronic dialysis patients whose plasma intact PTH levels were greater than 300 pg/ml were analyzed for the study. Following a four-week washout time during which all vitamin D administration was halted, 10 microg of maxacalcitol was intravenously injected thrice a week. RESULTS: The circulating intact PTH, bone-specific alkaline phosphatase and intact osteocalcin levels were significantly lowered, while the serum calcium levels were elevated after the therapy. The osteoprotegerin levels significantly decreased after the therapy (p < 0.0001). CONCLUSION: Maxacalcitol therapy reduced the circulating osteoprotegerin levels and improved secondary hyperparathyroidism. The observed effects were the opposite of those expected from previous in vitro studies. Osteoprotegerin may mediate and/or modify the effect of active vitamin D therapy in dialysis patients.

Calcitriol↗
PubMed2003

Peroxisome proliferator-activated receptor gamma C161T polymorphisms and survival of Japanese patients with immunoglobulin A nephropathy.

Peroxisome proliferator-activated receptor gamma (PPAR gamma) plays an important role in lipid metabolism, insulin sensitivity, atherogenesis, and immune regulation. A genetic polymorphism (C161T) at exon 6 of PPAR gamma gene (PPARG) was reported to be associated with the onset of coronary artery disease. However, there has been no report of an association with renal disease. Genomic DNAs were isolated from 225 Japanese patients with histologically confirmed immunoglobulin A nephropathy (IgAN). The PPARG C161T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism. The association of the polymorphism with renal prognosis in IgAN patients was analyzed using the Kaplan-Meier method and Cox proportional hazard regression model. The PPARG polymorphism was not associated with the renal survival rate. However, when patients were stratified into those either with or without hypertension at the time of diagnosis, the renal survival of the CT/TT genotypes was significantly better in those without hypertension than those with the CC genotype. We report that the PPARG C161T polymorphism is associated with the survival of IgAN patients without hypertension. The T allele of the polymorphism might have a protective effect on the progression of IgAN.

Adult↗
PubMed2002

Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors.

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Substitution of Dmo1 with normal alleles results in decreased manifestation of diabetes in OLETF rats.

AIM: Dmo1 (Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes in the Otsuka Long Evans Tokushima Fatty (OLETF) rat strain. To evaluate possible metabolic and pathological improvements generated by correction of the Dmo1 genetic pathway, we produced congenic lines, in which both OLETF Dmo1 alleles are replaced by the F344-derived genome. METHODS: Congenic animals were produced by introgressing F344-derived Dmo1 alleles into the OLETF rat. Congenic animals of the fourth generation (BC4) were intercrossed to obtain F1 animals (BC4:F1). Animals of the next generation, BC4:F2, were used for this study. We used 23 BC4:F2 males harbouring homozygous replacement of the OLETF Dmo1 region with the F344-derived genome. Seven animals with OLETF-derived Dmo1 alleles were used as controls. RESULTS: Dmo1-F344/F344 congenic rats showed significant decreases in body weight, abdominal fat weight, serum triacylglycerols, total cholesterol, food consumption and blood glucose after glucose loading (13%, 39%, 45%, 27%, 18% and 27% respectively; p < 0.05) compared with Dmo1-OLETF/OLETF animals. Furthermore, histopathological analysis of the kidney showed that mesangial sclerosis, hyalin deposits and deposition of PAS-positive substance were significantly lower in Dmo1-F344/F344 animals (p < 0.05). CONCLUSION: Improvements in metabolic parameters and histopathological scores show that correction of the Dmo1 genetic pathway in the diabetic and mildly obese OLETF rat strain produces wide-ranging therapeutic effects. Thus, this pathway might represent a new drug target also applicable to humans.

Animals↗
PubMed2001

Utilization of ATP-binding cassette exporter for hyperproduction of an exoprotein: construction of lipase-hyperproducing recombinant strains of Serratia marcescens.

The Serratia marcescens extracellular lipase (LipA) is an enzyme applicable to enantioselective hydrolysis of racemic substrates. The enzyme is secreted through an ATP-binding cassette (ABC) exporter, the Lip system, encoded by the lipBCD genes. The S. marcescens recombinant carrying pLIPE121, which encodes the lipA gene in pUC19, exhibited a higher LipA production level than the wild-type strain. However, the level was lower than expected, and secretion was suggested to be a bottleneck. lipBCD plasmids were introduced into S. marcescens recombinants harboring lipA plasmids and the effectiveness of the lipBCD plasmids in elevating LipA productivity was investigated. S. marcescens strains harboring both lipA and lipBCD plasmids showed sevenfold greater extracellular LipA activity than the strain harboring the lipA plasmid alone. A high level of extracellular LipA production (1,300 kU/ml) and high plasmid stability (enough to carry out large-scale cultivation) were observed under non-selective conditions. Addition of L-proline and Tween 80 was effective in increasing cell growth of the recombinant, which led to high LipA production. In batch cultivation using a 30-l jar fermentor, LipA production was achieved at a high level of 5,200 kU/ml. This is the first report describing utilization of ABC exporter for the overproduction of an industrially important extracellular protein.

ATP-Binding Cassette Transporters↗
PubMed2001

Molecular cloning and characterization of the Cl(-) pump-associated 55-kDa protein in rat brain.

The Cl(-)-ATPase/pump in the plasma membrane of the rat brain is a candidate for active outwardly directed Cl(-) translocating systems. We recently isolated a Cl(-) pump, 520- or 580-kDa protein complex, which consisted of 51-, 55-, 60-, and 62-kDa proteins. In this study, we cloned a cDNA encoding a 55-kDa glycoprotein, designated as ClP55, which contained an open reading frame of 1512 base pairs encoding a protein of 504 amino acids including a signal peptide of 28 amino acids. Northern and Western blot analyses demonstrated expression of ClP55 mainly in the cerebrum. Application of antisense phosphorothioate oligonucleotides to cultured neurons resulted in a marked increase in the intracellular Cl(-) concentration ([Cl(-)](i)). Immunohistochemical analysis indicated that ClP55 was localized to the plasma membranes of neurons such as hippocampal pyramidal neurons and cerebellar Purkinje cells. Taken together, these results suggest that ClP55 is one of the Cl(-) pump subunits responsible for Cl(-) pump activity.

Adenosine Triphosphatases↗
PubMed2001

Novel, potent, and selective phosphodiesterase 5 inhibitors: synthesis and biological activities of a series of 4-aryl-1-isoquinolinone derivatives.

A novel class of potent and selective phosphodiesterase 5 (PDE5) inhibitors, 4-aryl-1-isoquinolinone derivatives, which have been designed by the comparison of the structure of cGMP and a previously reported 1-arylnaphthalene lignan, was disclosed. Among these compounds, methyl 2-(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-3-isoquinoline carboxylate dihydrochloride (36a) exhibited potent PDE5 inhibitory activity (IC(50) = 1.0 nM) with high isozyme selectivities (IC(50) ratio: PDE1/PDE5 = 1300, PDE2/PDE5 > 10 000, PDE3/PDE5 > 10 000, PDE4/PDE5 = 4700, PDE6/PDE5 = 28). Compound 36a also showed the most potent relaxant effect on isolated rabbit corpus cavernosum (EC(30) = 7.9 nM). Compound 63 (T-1032), the sulfate form of 36a, was selected for further biological and pharmacological evaluation of erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2001

Serratia ATP-binding cassette protein exporter, Lip, recognizes a protein region upstream of the C terminus for specific secretion.

Serratia marcescens ATP-binding cassette (ABC) exporter, the Lip system, secretes lipase (LipA(SM)), metalloproteases, and a cell surface layer protein homologue but not a heme acquisition protein, HasA (HasA(SM)). Secretion of HasA(SM) is limited to the Has(SM) system. However, HasA proteins from Pseudomonas fluorescens (HasA(PF)) and Pseudomonas aeruginosa were exported through the Lip and Has(SM) systems. To investigate the specificity in Lip exporter-mediated secretion, secretion analysis was performed using chimeras containing the HasA(PF) and HasA(SM) sequences. The segment Val-Ala-Leu (designated R1 to R3 sites), which is present close to the C terminus of HasA(PF) but not HasA(SM), was revealed to be involved in the substrate specificity of the Lip exporter. Introduction of amino acid substitutions into the R1-R5 region demonstrated that R1, R3, R4, and R5 sites require some specific amino acid residues for Lip-mediated secretion. The amino acid sequence of the region was conserved considerably among the proteins secreted by the Lip exporter. On the contrary, the region was not related to HasA secretion through the Has(SM) system. Interestingly, a typical C-terminal motif, so far regarded as a secretion signal, was not necessary for secretion through either the Lip or the Has(SM) exporter. In LipA(SM) secretion via the Lip system, the typical C-terminal motif was not essential either, but the presence of a sequence similar to Val-Ala-Leu and its location from the C terminus greatly affect the secretion level. Secretion analyses using hybrid exporters and competitors exhibited that the R1-R5 region was recognized by an ABC protein of the Lip exporter, LipB, and that the mutations aborting Lip-mediated secretion in the region resulted in a loss of the affinity to LipB. Thus, a determinant within the secretory protein for Lip-mediated secretion was fully defined.

Amino Acid Sequence↗
PubMed2001

Developmental changes in Cl(-)-ATPase activity in rat brains.

Developmental changes in brain Cl(-)-ATPase activity were examined using fetal, neonatal and adult rats. The Cl(-)-ATPase activity rapidly increased over 20 postnatal days to a level four-fold higher than that in an 18-day-old fetus. On Western blot analysis using an anti-Cl(-)-ATPase/pump 51 kDa subunit (ClP51) antibody, the amount of ClP51 protein increased in parallel with Cl(-)-ATPase activity. Immunohistochemistry using the same antibody showed Cl(-)-ATPase-like immunoreactivity on the cell membranes of neurons such as cerebral and hippocampal pyramidal cells and cerebellar Purkinje cells, where the immunoreactivity increased with developmental changes in the size and shape of the neurons. These findings suggest that neuronal Cl(-)-ATPase activity markedly increases during early postnatal development with an increase in the amount of Cl(-)-ATPase protein, which may support the formation of inwardly directed neuronal Cl(-) gradients.

Adenosine Triphosphatases↗
PubMed2001

Expression of TMEFF1 mRNA in the mouse central nervous system: precise examination and comparative studies of TMEFF1 and TMEFF2.

TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and two follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebral neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated differently and that they play region-specific roles in the central nervous system.

Animals↗
PubMed2001

Human Ca2+/calmodulin-dependent phosphodiesterase PDE1A: novel splice variants, their specific expression, genomic organization, and chromosomal localization.

We report here the identification of novel human PDE1A splice variants, their tissue distribution patterns, genomic structure, and chromosomal localization of the gene. We identified one N-terminus (N3) and one C-terminus (C3) by cDNA library screening and dbEST database search. These N- and C-termini, including the reported N-termini (N1 and N2) and C-termini (C1 and C2), combined to generate nine different PDE1A cDNAs. N1 and N2 are similar to the 5' ends of the bovine PDE1A proteins of 61 kDa and 59 kDa, respectively, and C1 and C2 are the 3' ends of the reported human PDE1A variants. The results of PCR and Southern blot analysis show that nine PDE1A splice variants exhibit distinctive tissue distribution patterns by the difference of the N-terminus. PDE1As with N2 were widely expressed in various tissues, mainly in the kidney, liver, and pancreas. On the other hand, PDE1As with N1 and N3 were particularly expressed at a high level in the brain and testis, respectively. These findings suggest that the distinct expression patterns among PDE1A variants depend on the several promoters situated upstream of exons encoding 5' ends of the variants. The PDE1A gene spans over 120 kb of genomic DNA, and consists of at least 17 exons and 16 introns. The PDE1A gene was located on human chromosome 2q32 by fluorescent in situ hybridization analysis.

Amino Acid Sequence↗